Treatment of giardiasis and trichomoniasis

ABSTRACT

Fluorouracil and emimycin are useful in the treatment of giardiasis and trichomoniasis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of my co-pendingapplication Ser. No. 347,655 filed Feb. 10, 1982.

BACKGROUND OF THE INVENTION

Giardia lamblia, a parasite that until recently was widely believed tobe relatively harmless, now heads the list of the most common intestinalparasitic infections in the United States and some other countries. Theparasite causes a diarrheal disease call giardiasis.

This infection causes a variety of intestinal symptoms, such asprolonged diarrhea, abdominal cramps, stomach pain, severe weight loss,fatigue, nausea and flatulence.

Giardiasis can also cause malabsorption of nutrients and even retardedgrowth. Furthermore, giardiasis can mimic the symptoms of otherconditions such as ulcers and gall bladder attacks. If misdiagnosed, apatient may have a series of costly, needless tests, and even surgery.

The infection can be successfully treated with one of three drugs:Atabrine, Flagyl or furazolidione. However, each of these drugs is knownto cause adverse side effects. Until the present invention noprophylactic drug has been found which can adequately protect againstgiardiasis. (L. K. Altman, M.D., The New York Times, June 10, 1980).

Trichomoniasis is an infection of the lower genito-urinary tract, whichmay be induced in men and women by the protozoan parasite Trichomonasvaginalis. The infection may produce a few symptoms of such extremediscomfort and morbidity that intervention from a gynecologist or aurologist is necessary. The disease is of cosmopolitan distribution andapparently 10-25% of sexually mature females and 25-80% of theirconsorts are involved (E. A. Steck, The Chemotherapy of ProtozoanDiseases, Vol. II, Section 3, 17-1 (1971). Trichomoniasis is presentlytreated with Flagyl (metronidazole).

The present invention relates to the use of5-fluoro-2,4-(1H,3H)pyrimidenedione (fluorouracil) or2-hydroxypyrazine-4-oxide (emimycin) in the treatment of giardiasis andtrichomoniasis in humans.

SUMMARY OF THE INVENTION

The present invention is directed to the novel method for control andtreatment of giardiasis, a parasitic infection in humans caused byprotozoa of the genus Giardia.

The novel compositions used in the present method can also be used forthe prevention, treatment and control of trichomoniasis in men andwomen.

Therefore, it is an object of the present invention to (1) provide novelpharmaceutical composition comprising fluorouracil or emimycin withanti-giardiasis and anti-trichomoniasis activities; (2) provide a novelmethod for the prevention, control and/or treatment of giardiasis andtrichomoniasis in humans through the administration of the novelcompositions.

DETAILED DESCRIPTION OF THE INVENTION

The compounds useful in the novel methods of treatment of this inventionhave the following structural formulae: ##STR1##

The activity of these compounds against Giardia lamblia and Trichomonasfoetus are shown by the following test:

About 1.0-2.0 ml of a nutrient medium, for example, the modifiedDiamond's TPS medium at pH 7.05, together with about 10% by volume ofheat-inactiviated serum and about 1% by volume of antibiotic-antimycoticsolution is placed in each well of a multiwell plate. To this mixture,an aliquot of a suspension of G. lamblia cells containing about 10⁶organisms is added. Subsequently, each well is inoculated with a knownconcentration of one of the active compounds. The multiwell platecontaining the individual culture samples is incubated under anaerobicconditions at about 37° C. for about 16-24 hrs. The number of viablecells remaining in each well are then counted, such as with ahemacytometer. The percentage of survival is determined by comparisonwith controls inoculated with DMSO (dimethylsulfoxide) and the effectiveconcentration (in parts per million) for 50% inhibition of growth (ED₅₀)is determined. It is established that the lower the number of the valueof ED₅₀, the higher the activity of the active compound. The ED₅₀ valuesof the active compounds are summarized below in Table I.

                  TABLE 1                                                         ______________________________________                                        In vitro Anti-G. lamblia and anti-T. foetus                                   activities of fluoruracil and emimycin                                                       Anti-G.    Anti-T.                                                            lamblia    foetus                                              Compound       ED.sub.50 (ppm)                                                                          ED.sub.50 (ppm)                                     ______________________________________                                        (1) fluorouracil                                                                             100        3                                                   (2) emimycin   22.0       <1.0                                                ______________________________________                                    

The novel method of this invention comprises the administration offluorouracil or emimycin as an anti-giardiasis or anti-trichomoniasisagent to a human patient in amounts ranging from about 0.05 to about 50mg. per kg. of body weight, preferably from about 0.25 to about 25 mg.per kg. of body weight in a single dose or in 2 to 4 divided doses.

These compounds in the described dosages are usually administeredorally. They may also be administered to individuals by injection. Theoral phamaceutical compositions of this invention usually consist of anactive compound and some appropriate excipients and used in the form oftablets, troches, capsules, elixirs, suspensions, syrups, wafers,chewing gum and the like. The amount of an acitive compound in such atherapeutically useful composition or preparation usually ranges fromabout 2.5 mg. to about 2.5 g. preferably from about 5 mg. to about 500mg. per unit dosage.

The previously described tablets, troches, capsules, pills and the likeusually contain one or more of the following inactive ingredients: abinder such as gum tragacanth, acacia, corn starch or gelatin; anexcipient such as dicalcium phosphate; a disintegrating agent such ascorn starch, potato starch, alginic acid and the like; a lubricant suchas magnesium stearate; and a sweetening agent such as sucrose, lactoseor saccharin and/or a flavoring agent such as peppermint, oil ofwintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain in addition to materials of the above type, aliquid carrier such as fatty oil. Various other materials may be presentas coatings or to otherwise modify the physical form of the dosage unit,for instance, tablets, pills, or capsules may be coated with shellac,sugar, or both. A syrup or elixir may contain the active compounds,sucrose as a sweetening agent, methyl and propyl parabens aspreservatives, a dye and a flavoring such as cherry or orange flavor. Ofcourse, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially nontoxic in the amountsemployed.

EXAMPLE 1

In each well of a multiwell plate is placed 1.4 ml of media mixcontaining Diamond's TPS medium (see Table 2) at pH 7.05, 10% by volumeof heat-inactivated fetal bovine serum, and 1% by volume of anantibiotic-antimycotic solution (see Table 3). A suspension of Giardialamblia is centrifuged at 2,500 xg. for 6 minutes. The cells areresuspended in a small volume of the Diamond's medium, counted, and eachwell inoculated with approximately 10⁶ organisms. A stock solution offluorouracil is made at a concentration of 150 μg/ml. The wells are theninoculated with various concentrations of the drug. The plates areincubated for 24 hours at 37° C. in an anaerobic Gas Pak jar. After 24hours of incubation each well is mixed and counted for viable organismsusing a hemacytometer. The percentage of survival is determined bycomparing the treated wells to controls treated with dimethylsulfoxide.The in vitro anti-G. lamblia activity of fluorouracil is determined interms of ED₅₀ (effective dosage for 50% inhibition of growth) and has aED₅₀ value of 100 ppm.

                  TABLE 2                                                         ______________________________________                                        Composition of Diamond's TPS Medium                                           Ingredients             Amounts                                               ______________________________________                                        Trypticase (BBL)        1.00   g                                              Panmede, liver digest P & B                                                                           2.00   g                                              Glucose                 0.50   g                                              L-cysteine monohydrochloride                                                                          0.10   g                                              Ascorbic acid           0.02   g                                              Sodium chloride         0.05   g                                              Potassium phosphate-monobasic                                                                         0.06   g                                              Potassium phosphate, dibasic                                                                          0.10   g                                              anhydrous                                                                     Water, glass distilled  87.50  ml                                             pH adjusted to 7.0 with                                                       1 N NaOH                                                                      ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Composition of antibiotic-antimycotic solution (100X)                         ______________________________________                                        Penicillin,         10,000  units                                             Streptomycin        10,000  mcg.                                              Fungizone ®     25      mcg.                                              Prepared in 1 ml. of normal saline.                                           ______________________________________                                    

Employing substantially the similar procedure as described in Example 1but substituting for fluorouracil used therein the other active compoundemimycin, there is obtained similar results indicating the anti-G.lamblia activities of the compounds included above in Table 1.

Similarly, following essentially the same procedure described above, thehydrochloride salts of the compounds are found to be equally active asthe corresponding free base.

EXAMPLE 2

Employing essentially the same method of Example 1, centrifuged cells ofT. foetus KV₁ are inoculated with fluorouracil at 37° C. for 24 hours.The results indicate that this compound is also an effective anti-T.foetus agent with an ED₅₀ value of 3 ppm.

Similarly, emimycin and the pharmaceutically acceptable salts thereofare active anti-T. vaginalis agents as shown in Table 1.

EXAMPLE 3

    ______________________________________                                        Preparation of Capsule Formulation                                                            Milligrams per                                                Ingredient      Capsule                                                       ______________________________________                                        Fluorouracil    10                                                            Starch          100                                                           Magnesium Stearate                                                                            10                                                            ______________________________________                                    

The active ingredient, starch and magnesium stearate are blendedtogether. The mixture is used to fill hard shell gelatine capsules of asuitable size at a fill weight of 120 mg per capsule.

EXAMPLE 4

    ______________________________________                                        Preparation of Tablet Formulation                                                              Milligrams per                                               Ingredient       Capsule                                                      ______________________________________                                        Emimycin         12                                                           Lactose          200                                                          Corn starch (for mix)                                                                          50                                                           Magnesium stearate                                                                              6                                                           ______________________________________                                    

The active ingredient, lactose and corn starch (for mix) are blendedtogether. The corn starch (for paste) is suspended in water at a ratioof 10 grams of corn starch per 80 milliliters of water and heated withstirring to form a paste. This paste is then used to granulate the mixedpowders. The wet granules are passed through a No. 8 screen and dried at120° F. The dry granules are passed through a No. 16 screen. The mixtureis lubricated with magnesium stearate and compressed into tablets in asuitable tableting machine. Each tablet contains 12 milligrams of theactive ingredient.

What is claimed is:
 1. A method of treating giardiasis andtrichomoniasis in a human which comprises the administration to a humanin need of said treatment of an effective antigiardiasis andantitrichomoniasis amount of 2-hydroxypyrazine-4-oxide.